Atherosclerosis is the primary cause of cardiovascular disease, which is the most common cause of death in the United States. Atherosclerosis is characterized by the accumulation of lipids, cholesterol, calcium deposits, and cellular debris in vessel walls, and results in plaque formation, arterial obstruction, and diminished blood flow to organs. These plaques often rupture, causing myocardial infarction, stroke, or death. The main risk factors include elevated lipid levels, hypertension, and diabetes. Current treatment strategies are directed at changing patient lifestyle/diet and decreasing cholesterol via pharmacological methods. Surgical interventions with medical devices such as stents are used for advanced cases. While these therapeutic approaches have benefited many patients with this disease, they are far from ideal. One reason is that no drug or device is actually developed and tested in a model system that accurately recreates the disease being treated. The lack of an animal model that accurately replicates all of the manifestations of human atherosclerosis has been a major barrier to the development of effective therapies for this deadly disease. Several mouse models have been generated with mutations in genes important for lipoprotein metabolism, and while these models have been informative, they fail to develop the complex atherosclerotic lesions that are typical of the human disease. In contrast to mice, the physiology and anatomy of the porcine cardiovascular system closely resembles that of humans. In fact, pigs have long been used as models of cardiovascular disease, and pigs with naturally occurring mutations in their LDL receptor (LDLR) gene, and therefore possessing elevated LDL, have been reported. Although the hypercholesterolemic pig is an attractive model, the mild nature of the mutation, the high variability of the disease and the limited access by other researchers prevents its wide use in the research community. Therefore, the ultimate goal of this project is to develop and commercialize a gene- targeted porcine model of atherosclerosis. This proposal specifically outlines the development of porcine fibroblasts with a disrupted LDLR gene. A gene targeting vector will be constructed to disrupt the coding region of LDLR. Pig fetal fibroblasts will be infected with a virus carrying the LDLR targeting vector. Our plans for generating properly targeted cells are designed to maximize the frequency of homologous recombination, minimize random integration, and minimize the number of cell passages before targeted cells are harvested. Subsequent work will use these cells for somatic cell nuclear transfer to produce a porcine model of atherosclerosis. This animal model will provide academic and industrial research communities with an opportunity to better understand the disease and its pathogenesis and to develop and test new therapeutics and preventative strategies. PUBLIC HEALTH RELEVANCE: This proposal specifically outlines the development of porcine fibroblasts with a disrupted LDLR gene as a first step towards a new model of atherosclerosis. Subsequent work will use these cells for somatic cell nuclear transfer to produce pigs with elevated cholesterol. This animal model will provide academic and industrial research communities with an opportunity to better understand cardiovascular disease and its pathogenesis, and to develop and test new therapeutics and preventative strategies.